Alan. A. Liss, Inc. Receptor-Mediated. Endocytosis of Carcinoembryonic. Antigen by Rat Alveolar Macrophages. In Vitro. Carol Ann Toth, Alan Rapoza,. Norman.
Journal
of Leukocyte
Biology
45:370-376
(1989)
Receptor-Mediated Endocytosis of Carcinoembryonic Antigen by Rat Alveolar Macrophages In Vitro Carol
Ann
Laboratory
Mallory
Toth,
Alan
of Cancer
Rapoza,
Norman
Zamcheck,
Glenn
Steele,
and
Peter
Thomas
Biology, Department
Gastrointestinal
Research
G.S., PT.),
of Surgery, New England Deaconess Hospital (C.A.T., A.R., G.S., PT.), Laboratory, Boston City Hospital, (N.Z.), and Departments of Surgery (C.A.T., and Medicine, (N.Z.), Harvard Medical School, Boston
Uptake of carcinoembryonic antigen (CEA) by isolated rat alveolar cells was time, temperature, and concentration dependent (KUP.k. = 2.4 x 1 01M). Pretreatment of the alveolar cells with colchicine inhibited internalization of CEA. Uptake of 1251-labeled CEA by alveolar cells requIred divalent catlons and was inhibited by cold CEA and nonspecific cross-reacting antigen (NCA). The carbohydrate portion of CEA was modified by neuraminidase treatment and Smith degradation. The modified glycoproteins inhibited
endocytosis by the alveolar macrophages, thus excluding nonreducing terminal carbohydrate residues as the recognition site of the receptor. Endocytosis of CEA was independent of native protein conformation since performic acid oxidized CEA and glycopeptides produced by pepsin digestion were inhibitory. Rat alveolar macrophages bound CEA with similar specificity to that of rat Kupffer cells. Alveolar macrophages, unlike Kupifer macrophage-like
endocytosing Key words:
cells, did not rapidly exocytose murine cell line, nor murine
nonspecific
cross-reacting
antigen,
Carcinoembryonic antigen (CEA) is a 180-kD glycoprotein secreted by and present on the cell surfaces of various adenocarcinomas [27]. Serial determinations of plasma levels of this tumor-associated antigen serve as an indicator of prognosis and as a monitor of therapy for carcinoma of the colon, breast, lung, and pancreas [16,26]. CEA is composed of a single polypeptide chain with three 178-residue repetitive domains, each containing two disulphide bridges attached to a 108-residue amino terminal domain and a small hydrophobic Cterminal fragment [8]. The disulphide bridge domains share an exceptionally high level of sequence homology [28]. The amino terminal domain does not contain cystemne; however, it exhibits extensive homology with the disulphide bridge domains [17]. Complex carbohydrate chains compose over 50% of the molecule by weight and are linked to 28 asparagine residues largely on the disuiphide bridge domains [3]. Biochemical and immunological studies have shown that CEA is 1 of 6-10 molecules of a gene family which are expressed in different tissues at various times [17]. Nonspecific cross-reacting antigen (NCA), a related molecule, shares 89% sequence homology with CEA [17]. The disulphide bridge domains of the CEA-related molecules share remarkable sequence homology with Alan
A. Liss, Inc.
a of
CEA in vitro.
INTRODUCTION
© 1989
the internalized CEA. Neither P38801, peritoneal exudate cells were capable
Kupifer
cell,
P388D1
members of the immunoglobulin supergene family, in particular mouse Thy 1 antigen, K-chain-variable regions, rabbit polyimmunoglobulmn receptor, and human secretory component [9,10]. CEA and NCA are primarily removed from the circulation by the Kupffer cells of the liver but significant amounts are also taken up by the spleen and lungs [15]. In the absence of a functional liver the major organ of uptake becomes the lungs. CEA and NCA are bound and endocytosed by the same receptor system on the rat Kupffer cell [18,19]. In the human, alveolar macrophages and Kupffer cells have NCA present normally on their surfaces [2,6], and isolated human Kupffer cells can endocytose both CEA and NCA [20]. This paper describes the ability of macrophage populations other than Kupffer cells to endocytose CEA in vitro.
MATERIALS CEA CEA
was
AND METHODS isolated
tal adenocarcinomas
Received Reprint Laboratory
August
from
hepatic
by perchloric
23, 1988; accepted
metastasis acid
November
of colorecextraction
and
21, 1988.
requests: Carol A. Toth, New England Deaconess Hospital, of Cancer Biology, 50 Binney Street, Boston, MA 02115.
Endocytosis
of CEA by Alveolar
M#{216} 371
purification by Sepharose 4B and Sephadex G-200 chromatography [22]. The resulting preparation ran as a single 180-kD band on SDS-PAGE and as a single peak on a TSK-4000 HPLC gel filtration column. The preparation was equivalent in activity in to the Roche standard CEA by radioimmunoassay (Roche, Nutley, NJ).
Radiolabeling
Chemical
Alveolar macrophages were isolated from the lungs of unstimulated male rats (Sprague Dawley 250 g) by lavage with PBS without Mg2 and Ca2. The lavage fluid was centrifuged and the resulting cell pellet was washed in Minimal Essential Medium (MEM) containing BSA (1%). Eighty-six percent of the alveolar cells stained positively for nonspecific esterase activity [5] and 80% phagocytosed colloidal carbon. Experimental cell populations were greater than 95% viable by trypan blue dye exclusion.
Modifications
of CEA
Asialo CEA was produced by neuraminidase digestion of CEA [1]. Smith-degraded CEA was produced by sequential sodium periodate oxidation, sodium borohydride reduction, and mild acid hydrolysis as previously described [1]. The disuiphide bridges on CEA were oxidized by performic acid treatment [25]. Limited enzymatic cleavage of CEA with pepsin (Sigma) was performed as previously described [23] and the resulting glycopeptides were separated by HPLC on a TSK-3000 gel filtration column.
Nonspecific
Cross-Reacting
Antigen
NCA was isolated from a liver metastasis of colonic adenocarcinoma by perchioric acid extraction and chromatography on Sepharose 4B and Sephadex G-200 columns. Final purification was by DEAE cellulose chromatography and acetone precipitation [24]. The resulting NCA ran as a single 54-kD band on SDS-PAGE and as a single peak on a HPLC TSK-3000 column. The purification of the glycoprotein was monitored by radioimmunoassay [24].
Alpha1acid
Glycoprotein
Alpha1acid glycoprotein was purified from pooled human serum by DEAE cellulose chromatography and ammonium sulphate precipitation [19]. Isolated alphaiacid glycoprotein ran as a single 44-kD band on SDS-PAGE. Asialo alphaiacid glycoprotein was produced by neuraminidase treatment of the native glycoprotein.
of Glycoproteins
Glycoproteins (New
England
Alveolar
Murine
were
labeled
with
Nuclear)
by using
Macrophage
Isolation
sodium chloramine-T
iodide
(125!)
[18].
Cell Line P38801
P388D1, a murine macrophage-like cell line, was maintained in RPMI 1640 (10% FCS) with L-glutamine. Experimental cell populations were greater than 95% viable and 99% of the viable cells phagocytosed colloidal carbon.
Peritoneal
Exudate
Cell Isolation
Peritoneal exudate cells were isolated from female CD-i mice (Charles River Breeding Lab) by pentoneal lavage with MEM. The lavage fluid was centrifuged and the resulting cell pellet was washed with MEM (1% BSA). The isolated cells were greater than 90% viable and composed of 20-30% macrophages as determined by nonspecific esterase staining [5] and phagocytosis of colloidal carbon. Since these cells are a less-pure population of macrophages than either the alveolar or P388D1 cells, cell numbers were adjusted so that approximately the same number of macrophages were present in each assay.
Other
Glycoproteins
Fetuin was purchased from Sigma Chemical Louis, MO) and desialylated with neuraminidase. mannan was purchased from Sigma.
Carbohydrate
Co.
(St. Yeast
Analysis
Total carbohydrate analysis of glycoproteins was performed by the method of Reinhold [11]. Sialic acid content of the glycoproteins was determined by the thiobarbituric acid assay [21] or gas liquid chromatography [11].
Circular
Dichroism
Analysis
The secondary structure of CEA was determined by using an AVIV 60 DS CD Spectropolorimeter and the resulting data were analyzed by the Yang equation [4].
Determination Cells
of Glycoprotein
Uptake
by Isolated
Glycoprotein uptake by macrophages was measured by incubating isolated cells with radiolabeled ligand (7.5 pg/ml) in buffered MEM (1% BSA) [12]; each assay tube contained approximately 2.0 x 106 macrophages. The incubation mixture was sampled in duplicate at various times and the cells were separated from the incubation medium by centrifugation at 11 ,000g for 5 mm through an oil phase (silicone and mineral oils, 4:1). The tip of the microcentrifuge tube containing the cell pellet was cut off and the 1251 content of both the supernatant and the cell fraction was determined by scintillation counting.
372
Toth et al tubule formation and receptor-mediated endocytosis [7], resulted in a 42% inhibition of CEA uptake but did not affect cell viability. Cellular uptake of CEA by alveolar
‘5
macrophages
addition inhibited
required
the
presence
of divalent
cations;
of EDTA (10 mM) to the incubation medium CEA uptake by 100%, without affecting cell
viability.
U
0
F U
‘U
40
20 TIME
60
(mm.)
Fig. 1. #{149} Uptake of 125i-CEA 7.5 g/ml by isolated alveolar macrophages at 37#{176}C. #{149} Inhibition of the uptake of 125 i-CEA 7.5 pg/mi by a 53-fold molar excess of unlabeled CEA at 37#{176}C. A Uptake of’25 1-CEA 7.5 ig/ml medium at 4#{176}C. Data points are the averages of duplicate samples of 1.9 x 106 alveolar macrophages.
Various chemical and enzymatic modifications of CEA were tested for their ability to inhibit uptake of labeled CEA by isolated alveolar macrophages. Neuraminidase treatment was used to remove sialic acid from CEA exposing the penultimate galactose moieties; Table 1 shows the carbohydrate analysis of asialo CEA. In the presence of unlabeled asialo CEA endocytosis of ‘251-CEA was inhibited by 61% (Table 2). However CEA uptake was not affected by the presence of a 1,300-fold molar excess of unlabeled asialo alpha1acid glycoprotein or a 836-fold molar excess of asialo fetuin. The removal of terminal carbohydrate groups and those internal sugars containing vicinal diol groupings by the Smith degradation resulted in the loss of 70% of the total carbohydrate. Carbohydrate analysis of Smithdegraded CEA showed complete loss of fucose and sialic acid with substantial reduction in galactose (8 1%), mannose (72%), and N-acetyl-glucosamine (61%) (Table 1). After the Smith degradation CEA still retained the ability to inhibit endocytosis of labeled CEA by alveolar macrophages
Electrophoresis Radiolabeled samples were pretreated with mercaptoethanol and SDS and run on 7.5% polyacrylamide slab gels. The gels were fixed, stained, dried, and exposed to Kodak X-OMAT XR-5 film at -20#{176}C. Isoelectric
Focusing
Isoelectric focusing was performed in 0.5% agarose gels (ampholyte range 2.5-10.0; LKB). The gels were fixed, dried, and examined by autoradiography as above.
RESULTS Uptake of ‘251-labeled CEA by isolated alveolar macrophages was studied in suspension assay. In timedependent experiments the uptake of ‘25I-labeled CEA was saturable and could be inhibited by a 50-fold molar excess of unlabeled CEA (Fig. 1). Uptake was temperature (Fig. 1) and concentration dependent (Fig. 2). Analysis of the concentration-dependent data by a double reciprocal plot indicated an approximate Km of 2.4 x l0M (Fig. 2). Pretreatment of the alveolar macrophages for 60 mm at 37#{176}C with colchicine (75 mM), an inhibitor of micro-
(Table
2).
Modification of CEA by performic acid oxidation results in breakage of the 6 intrachain disulphide bridges accompanied by loss of secondary conformation [25]. Circular dichroism analysis of the performic-acidoxidized CEA showed a complete loss of beta turns, a reduction in beta sheet, and an increase in random coil structure when compared to native CEA. Performicacid-oxidized CEA ran on HPLC (TSK 4000) with a shorter retention time than untreated CEA, suggesting a more extended conformation. Perf orm ic-acid-oxidized CEA present in a 200-fold molar excess inhibited in vitro endocytosis
of
labeled
CEA
by
alveolar
macrophages
(Table 2). Limited pepsin digestion of CEA results in the production of six glycopeptides (7,000-16,000 M4). Endocytosis of 1251 CEA by alveolar macrophages was inhibited 38% in the presence of a 151-fold molar excess of unlabeled pepsin glycopeptides. Isolated alveolar macrophages ture produced
were by
incubated the pepsin
with the digestion
glycopeptide of CEA;
mixHPLC
analysis of the supernatants showed that the cells preferentially removed an I l-kD glycopeptide from the medium. Rat alveolar macrophages endocytose yeast mannan by a mannose/N-acetyl-glucosamine receptor [12]. In the cell suspension assay, alveolar macrophages took up
Endocytosis
of CEA by Alveolar
M#{216}
373
B A U S U U
C
f
0
S
a
S
U
a
0.
.6 ‘U
U
a
U
‘U
U 01
0 CEA
ug/mi
(CEa
ugAnl)
Fig. 2. CEA specific uptake by Isolated alveolar macrophages. A. Uptake of increasing concentrations of ‘251-CEA by alveolar macrophages was carried out for 30 mm at 37#{176}C by using the cell suspension assay. Data points are the average of duplicate
samples (1.6 x 106 cells). Nonspecific uptake was determined in the presence of 2 mg/mI of unlabeled CEA and subtracted from the totals. B. Double reciprocal plot. Km = 2.4 x 107M (r = .998).
TABLE 1. Carbohydrate Smith-Degraded CEA
TABLE
Analysis
of CEA, Asialo-CEA,
and
2. Inhibition
of CEA
Uptake
by Structurally
ModIfied
CEA
Carboh ydrate (% of total molecular
weight)
Smith-degraded CEA
Asialo-CEA
CEA
Fucose Mannose Galactose N-acetylglucosamine Neuraminic acid
13.0 10.6 13.2 26.4 5.7
10.2 8.0 9.9 19.7
0 2.9 10.2
0.5
0.3
Total carbohydrate
68.9
48.3
15.8
2.4
Inhibitor Asialo
Smith-degraded
CEA
Performic-acid-
oxidized
Determinations
excess
% inhibition
of control”
200-fold
60.7 (± 2.6)
200-fold
61.0
(±
3.7)
200-fold
53.3
(±
4.0)
CEA
aAll experiments
were performed of uptake
by using
of ‘I-labeled
the cell CEA
(7.5
106 cells were made after 60 mm at 37#{176}C. bData are presented as the average of six determinations. deviation
1251-labeled yeast mannan in a saturable and timedependent manner. Unlabeled mannan (78 pg/ml) inhibited uptake of labeled mannan (7.8 g/ml) by 70%. However, incubation with unlabeled yeast mannan (700 g/ml) failed to block uptake of labeled CEA (6.3 g/ml). NCA, a glycoprotein sharing structural similarities with CEA [17], is also able to inhibit endocytosis of CEA by alveolar macrophages (Fig. 3). However, NCA is not as effective an inhibitor of CEA endocytosis as CEA. In vitro a 925-fold molar excess of unlabeled NCA was needed to inhibit CEA uptake by 60%. Inhibition to the same degree by CEA only required the presence of a 50-fold molar excess. To determine if ligand was released from the cells after
Molar CEA
suspension g/ml)
assay. by
The
1.5
x
standard
is in parentheses.
uptake, the macrophages were incubated for 1 h with radiolabeled CEA (20 i.g/ml), washed repeatedly, and resuspended in CEA-free medium. The medium was then sampled at 10-mm intervals for 1 h, and the amount of released 1251 activity was determined. The cells released 20% of the total label taken up in 60 mm. The supernatants were analyzed by SDS-PAGE and agarose isoelectric focusing to determine the nature of the released activity. Neither SDS-PAGE nor agarose isoelectric focusing analysis demonstrated the release of labeled peptides larger than 10,000 Mr by the cells. A murine cell line (P388D1) derived from a methylcholanthrene-induced lymphoid neoplasm of a DBA/2 mouse has been reported to exhibit many of the functions
374
Toth et al
of the interaction with the macrophage. Because CEA has 28 possible sites for N-linked oligosaccharide chains, with the internal structure of the chains appearing the same with variations occurring at the terminal sugar residues and in the degree of mannose branching [3], the carbohydrate portion of CEA was modified by neuramina idase digestion and one cycle of the Smith degradation. U However, desialylated CEA and Smith-degraded CEA S both inhibited endocytosis of native CEA. This eliminates fucose, sialic acid, and other terminal carbohydrate groups as the site of receptor recognition. Previous aS studies with isolated rat Kupffer cells revealed the same insensitivity of receptor activity to carbohydrate modifications of CEA [19]. Since the Smith-degraded CEA still contains 16% carbohydrate by weight (mannose, N-acetylglucosamine, and galactose) there is a possibility that internal sugars are recognized. However, this seems unlikely unless the conformation of the carbohydrate chains on 10 12 0 2 4 the fully glycosylated molecule is such that internal GSA Cosic. .s,’. sugars are exposed. Alveolar macrophages also have a Fig. 3. Concentration-dependent uptake of CEA (‘l) in the receptor which binds glycoproteins with terminal manpresence of unlabeled NCA.. 1251-CEA uptake, 45 mm (37#{176}C); A nose or N-acetylglucosamine groups [12]. Inhibition 1251CEA uptake in the presence of unlabeled NCA (9.1 x studies using ligands for this receptor have shown that 106M). this is not the receptor which recognizes CEA. Studies on the secondary structure of CEA showed that it has a substantial amount of beta conformation with of normal macrophages but not to express mannose/Nlittle alpha helix and some random coil [25]. Modificaacetylglucosamine receptor activity [13]. When the cell tion of CEA by performic acid oxidation resulted in suspension assay was used, P388D1 cells did not take up breakage of intrachain disulphide bridges and loss of ‘251-labeled yeast mannan in a saturable manner. Simisecondary conformation. Inhibition of CEA uptake by larly, ‘251-CEA was not taken up by this cell line. both performic-acid-oxidized CEA and glycopeptides Peritoneal exudate cells were harvested from unstimfrom pepsin digestion indicated that receptor recognition ulated female mice by peritoneal lavage. In the cell is independent of native protein conformation. It is likely suspension assay these cells endocytosed radiolabeled that the receptor recognizes a specific random coil yeast mannan in a saturable and time-dependent manner sequence in the polypeptide chain. and phagocytosed microaggregated albumin. However, NCA is recognized by the same receptor as CEA on specific uptake of ‘251-CEA at 7.5 p.g/ml by the peritothe alveolar macrophage but has a lower affinity for the neal exudate cells could not be demonstrated. receptor. In vivo, NCA is cleared from the circulation by the same mechanism as CEA [18] with a half-life twice as long as that for CEA. In vitro, Kupffer cells endocyDISCUSSION tose NCA with a of 1.4 X l0 M compared to The saturability, concentration and temperature de6.0 x 10-8 M for CEA [19]. Consistent with the data presented here for alveolar macrophages, greater concenpendence, and colchicine sensitivity of CEA uptake by rat alveolar macrophages indicates that the process is by trations of NCA than CEA are required to inhibit receptor-mediated endocytosis. CEA uptake by alveolar circulatory clearance of CEA in vivo and by isolated macrophages has an apparent Km of 2.4 x l0 M and Kupffer cells [18,19]. Extensive internal protein serequires divalent cations. Previously published data quence homology (89%) exists between NCA and CEA showed that isolated Kupffer cells endocytose CEA with [10]. It is possible that the alveolar macrophage binds a an apparent Km of 6 x 108 M and have approximately protein sequence common to both glycoprotemns. While NCA and CEA are closely related, small structural 16,000 divalent cation-dependent receptor sites per cell [19]. changes around the macrophage-binding site could alter receptor affinity. Chemically and enzymatically modified CEA were used as inhibitors of endocytosis to determine the nature Following endocytosis in vitro the Kupffer cells re-
Endocytosis lease CEA with no apparent change in molecular weight but with a higher isoelectric point indicative of the removal of negatively charged moieties that are probably terminal sialic acid residues [19]. In vivo, the Kupffer cell transfers immunologically identifiable CEA to the hepatocyte where degradation is completed [18]. Under saturating conditions, alveolar cells appear to internalize 4 times the amount of labeled CEA as do isolated Kupffer cells. However unlike Kupffer cells, the alveolar cells do not exocytose the labeled CEA. Only the macrophages of the liver and lungs have been shown to carry this receptor system. Neither peritoneal macrophages nor the macrophage cell line P388D1 could be shown to endocytose CEA in a saturable manner. In the case of P388D1 this result is not surprising as the cells also do not express the mannose/N-acetyl glucosamine receptor system [13]. However, in the case of the peritoneal macrophages the absence of a CEA receptor system was not expected and is an example of a difference between macrophages that may have biological importance especially in the case of seeding of colorectal cancer cells to form metastases [14]. The differences between Kupffer cells and alveolar macrophages in the processing of endocytosed CEA may provide a model system in which to study, in one instance, (Kupffer cell) receptor-mediated uptake recycling and exocytosis where degradation of the ligand does not occur, and in the second case, (alveolar cells) where retention and degradation occurs in a more usual manner. The differences in handling of glycoprotein endocytosis by Kupffer cells and alveolar cells demonstrate that macrophages are a heterogeneous population of cells, exhibiting a degree of functional differentiation. Despite their common origin in the bone marrow tissue, macrophages tend to adapt to their specific anatomical location and exhibit characteristic differences in biochemical and functional parameters. The two major sites of metastatic spread for colorectal carcinoma are the liver and lungs; alveolar macrophages and Kupffer cells have similar receptors for CEA and NCA both of which are products of the malignant colonic epithelium. However, the precise nature of the interaction between immune regulatory cells and CEA remains to be elucidated.
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